Comparative Genomics and Regulatory Evolution: Conservation and
Function of the Chs and Apetala3 Promoters
Marcus A. Koch,* Bernd Weisshaar,† Juergen Kroymann,‡ Bernhard Haubold,‡
and Thomas Mitchell-Olds‡
*Department of Botany, University of Agricultural Science, Vienna, Austria; †Department of Biochemistry, Max-PlanckInstitute for Plant Breeding, Cologne, Germany; and ‡Department of Genetics and Evolution, Max-Planck-Institute for
Chemical Ecology, Jena, Germany
DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species
were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence
of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm
the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses,
indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory
elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic
trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic
relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the
phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates
the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action
and gene regulation.
Introduction
Improved understanding of gene regulation is essential for genomics and evolutionary biology. Within
species, regulatory pathways control development and
organismal responses to environment and may contribute to genetic variation for quantitative traits (Wang et
al. 1999) and disease (Horikawa et al. 2000; Maleck et
al. 2000). Among species, changes in gene regulation
may be fundamentally important for interspecific differentiation (Ting et al. 1998; Kopp, Duncan, and Carroll
2000). However, gene regulation is poorly understood,
especially in plants.
With the completion of Arabidopsis genomic sequencing and the new 2010 project to determine the
function of all Arabidopsis genes, there will be many
opportunities to understand gene regulation and its variation within and among species. Large-scale genome
annotation is routinely done automatically using database searches and gene prediction programs (Lin et al.
1999; Mayer et al. 1999). The latter are usually designed
to detect protein-coding regions, leaving the untranslated regulatory sequences of a gene largely unexplored.
It is well known that functionally important homologous
regions in coding sequences tend to be highly conserved
between sibling species. Similarly, it is expected that
regulatory elements in untranslated regions might be detected by sequence comparison (‘‘phylogenetic footprinting’’; Wasserman et al. 2000). Here, we test the
usefulness of this comparative strategy for the study of
Key words: Apetala3, Chs, comparative genomics, phylogeny,
promoter function, regulatory evolution.
Address for correspondence and reprints: Marcus A. Koch, Department of Botany, University of Agricultural Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria.
E-mail: [email protected].
Mol. Biol. Evol. 18(10):1882–1891. 2001
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1882
gene regulation using two genes, chalcone synthase
(Chs) and Apetela3, from 22 cruciferous species.
Recent studies have established a well-supported
overview of evolutionary relationships within the Brassicaceae (O’Kane and Al-Shehbaz 1997; Galloway,
Malmberg, and Price 1998; Koch, Bishop, and MitchellOlds 1999; Koch, Haubold, and Mitchell-Olds 2000,
2001). Divergence times among various taxa have been
estimated using several independent loci and fossil calibrations (Yang et al. 1999; Koch, Haubold, and Mitchell-Olds 2000). Consequently, these studies provide a
basis for selecting informative species for comparative
analysis and provide a key to the understanding of rates
and modes of regulatory evolution in the Brassicaceae.
Arabidopsis thaliana and many closely related species
are diploids with relatively few recent gene duplications.
This makes it comparatively easy to identify orthologous loci for comparative studies. However, complications may arise in ancient polyploids such as Brassica
(Kowalski et al. 1994; Lagercrantz 1998).
To obtain new insights into regulatory evolution,
we chose the well-characterized promoter regions of the
Chs and Apetala3 genes from selected species of the
Brassicaceae. Chalcone synthase participates in plant
secondary metabolism and catalyzes the key reaction in
flavonoid biosynthesis (Hahlbrock and Scheel 1989).
Although many plant species contain multiple Chs genes
(Ryder et al. 1987; Koes, Spelt, and van der Elzen 1989;
Wingender et al. 1989; An et al. 1993; Junghans, Dalkin,
and Dixon 1993; Durbin et al. 1995; Howles, Arioli, and
Weinman 1995), Chs is single-copy in A. thaliana (Burbulis, Iacobucci, and Shirley 1996) and most related diploids (Koch, Haubold, and Mitchell-Olds 2000). The
Chs promoter is responsive to UV light, and it has been
characterized experimentally in A. thaliana (Hartmann
et al. 1998) and Sinapis alba (Kaiser and Batschauer
1995; Kaiser et al. 1995). In addition, detailed infor-
Promoter Analysis in Cruciferous Plants
mation on the cis-acting elements and potential corresponding trans-acting factors of the Chs promoter region are available for many other plant species (Faktor
et al. 1997a; Feldbrügge et al. 1997; Seki et al. 1997
and references therein).
The floral homeotic gene Apetala3 is a well-known
MADS-box regulatory gene responsible for floral organ
identity. The function of this gene has been described
(e.g., Okamoto et al. 1994; Yi and Jack 1998), and its
promoter has been analyzed (Hill et al. 1998). The molecular evolution of the MADS-box genes including
Apetala3 has been studied in detail in higher plants (e.g.,
Purugganan et al. 1995; Kramer, Dorit, and Irish 1998;
Lawton-Rauh, Alvarez-Buylla, and Purugganan 2000;
Purugganan 2000), and phylogenetic data on Apetala3
from various Brassicaceae are available (Lawton-Rauh,
Buckler, and Purugganan 1999).
In this paper, we address three main questions: (1)
Can comparative analysis of promoter sequences among
related taxa identify conserved cis-acting regulatory elements? (2) Do conserved promoter elements confer
functional patterns of gene regulation? (3) Do patterns
of promoter evolution mirror known phylogenetic relationships inferred from nuclear genes, plastidic loci, and
noncoding spacer regions of nuclear ribosomal DNA?
Materials and Methods
A set of species from the Brassicaceae was selected
on the basis of previous phylogenetic relationships derived from different molecular markers. The various
markers analyzed were the internal transcribed spacer
(ITS) region of nuclear ribosomal DNA (Koch, Bishop,
and Mitchell-Olds 1999), the nuclear genes Chs and
ADH (Koch, Haubold, and Mitchell-Olds 2000), and the
plastidic matK gene (Koch, Haubold, and Mitchell-Olds
2001). Details on the species and accessions are given
in table 1. We investigated one individual per accession.
Alignments of Apetala3 and Chs promoter regions (supplementary material, figs. 1 and 2) are available via the
online version of the manuscript.
DNA Amplification, Cloning, and Sequencing
Total DNA was obtained from leaf tissue of single
individuals by a modified CTAB procedure (Mummenhoff and Koch 1994). Polymerase chain reaction (PCR)
was carried out using an ABI 9700 (Applied Biosystems). The PCR cycling scheme was 5 min at 958C; 35
cycles of 1 min at 958C, 1 min at 50–558C (depending
on primer combination), and 1 min at 728C; 15 min
extension at 728C; and a final hold at 48C. The oligonucleotide sequences used to amplify promoter fragments were as follows: Chs—CHS1.for (59-gagttaagtatgcacgtg-39) and CHS.rev (59-gagatcagaaggcacagag-39);
Apetala3—APET1.for (59-ggcttttaacaccaatataaaaa-39),
APET2.for (59-caatataaaaacttggttcacac-39), APET3.for
(59-gccaaccaaatccacctgca-39), and APET.rev (59-gagagggaagatccagatcaagagg-39). We designed several forward primers by comparing database sequences from
Brassica oleracea (Hill et al. 1998; AF043610) and A.
thaliana (Irish and Yamamoto 1995; U30729), because
1883
DNAs from several taxa did not amplify well with
APET1.for. The primer APET2.for overlapped with
APET1.for at its 59 end. DNAs which did not provide
good PCR results for primer APET1.for or APET2.for
originated from Cochlearia pyrenaica, Arabis alpina,
Matthiola incana, Arabis glabra, and Arabis turrita (table 1 and fig. 2, supplementary material). DNA extracted from these individuals was amplified using a third
primer, APET3.for. This primer is located 40 bp downstream of the APET2.for primer sequence in A. thaliana
and 485 bp downstream of the APET2.for primer sequence in B. oleracea. For an overview of the success
of different primer combinations in amplifying Apetala3
promoter regions, see table 2.
PCR reactions (50 ml) were performed under the
following conditions: 50 ng template DNA, 2 ng/ primer, 2.5 mM MgCl2, and 2 U Taq DNA polymerase. All
PCR products were purified from an agarose gel using
the Boehringer PCR product purification kit and cloned
either into pGEM-T cloning vector (Promega) or into
the TA kit pCR II cloning vector (Invitrogen). The PCR
products from all different Apetala3 amplifications (table 2) were cloned as well. For each DNA sample, we
performed two independent PCR reactions. From each
PCR reaction, two independently cloned PCR products
were sequenced separately to detect possible sequence
variation due to polymerase errors.
For each promoter fragment, both strands were cycle-sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI Applied Biosystem, Inc.).
Products of the cycle sequencing reactions were run on
an ABI 377XL automated sequencer (ABI Applied
Biosystem, Inc.). Cloned PCR products were sequenced
using universal t7 forward (59-gtaacgatttaggtgacactatcg39) and M13-48 reverse (59-agcggataacaatttcacacagga39) primers.
Subcloning of Chs Promoter Fragments and
Expression Analysis
Based on the Chs promoter sequences, primers
were designed which annealed to the ATG start codon
region and a position at about 2220 relative to the transcriptional start site. The upstream primer added a
HindIII site, and the downstream primer added a NcoI
site which was placed on the ATG. PCR fragments representing the various Chs promoter sequences were cut
with HindIII and NcoI and cloned into similarly digested
pBT10-GUS (Sprenger-Haussels and Weisshaar 2000).
Identities of clones were confirmed by sequencing. Expression analysis was performed as described (Hartmann
et al. 1998; Sprenger-Haussels and Weisshaar 2000) using a luciferase expression construct as an internal
control.
Phylogenetic Analysis
The sequence from S. alba was published by Batschauer, Ehmann, and Schafer (1991) under accession
number X17437. Promoter regions were aligned by
hand, and highly conserved regions served as anchor
regions for successive alignment. Only those parts of the
1884
Koch et al.
Table 1
Accession Data for Taxa Under Study
GENBANK ACCESSION NO.
TAXON, SYNONYMS, ORIGIN
Aethionema grandiflora L.
(Bot. Garden Jena, German) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Arabidopsis griffithiana Busch
(Olimarabidopsis pumila (Stephan) Al-Shehbaz, O’Kane, and
Price) (CS3701, ABRC, Ohio, USA) . . . . . . . . . . . . . . . . . . . .
Arabidopsis thaliana (L.) Heynh. [COL] . . . . . . . . . . . . . . . . . . .
A. thaliana (L.) Heynh. [LER] . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. thaliana (L.) Heynh.
(Hagen a.T.W., Lower Saxony, Germany) . . . . . . . . . . . . . . . . .
Arabis alpina L.
(Bot. Garden Bayreuth, Mt. Kenya, Kenya) . . . . . . . . . . . . . . .
A. alpina L.
(Pottenstein, Bavaria, Germany) . . . . . . . . . . . . . . . . . . . . . . . . .
Arabis drummondii A. Gray
5Boechera drummondii [Gray] Löve and Löve) (Mule
Creek, Mont., USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Arabis glabra (L.). Bernh. (5Turrits glabra L.)
(Niedersteinbeck, NRW, Germany) . . . . . . . . . . . . . . . . . . . . . . .
Arabis hirsuta (L.) Scop.
(Lengerich, NRW, Germany). . . . . . . . . . . . . . . . . . . . . . . . . . . .
Arabis jaquinii Beck
(Bot. Garden Jena, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . .
Arabis turrita L.
(Bot. Garden Berlin-Dahlem, Germany). . . . . . . . . . . . . . . . . . .
Barbarea vulgaris R. Br.
(Hasbergen, Lower Saxony, Germany). . . . . . . . . . . . . . . . . . . .
Brassica oleracea L. ssp. botrytis . . . . . . . . . . . . . . . . . . . . . . . . .
Capsella rubella Reuter
(no. 774, collection HURKA, Osnabrück, Germany) . . . . . . . .
Cardamine amara L.
(Hagen a.T.W., Lower Saxony, Germany) . . . . . . . . . . . . . . . . .
Cardaminopsis petraea (L.) Hil.
(5Arabidopsis lyrata [L.] ssp. petraea O’Kane and
Al-Shehbaz) (Plech, Bavaria, Germany). . . . . . . . . . . . . . . . . . .
Cardaminopsis halleri (L.) Hayek
(Arabidopsis halleri [L.] O’Kane and Al-Shehbaz)
(Blankenrode, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cochlearia excelsa J. Zahlbr. ex Fritsch
(Eisenhut, Turrach, Austria). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cochlearia pyrenaica L.
(Alme, NRW, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fourraea alpina (L.). Greut. et Burd.
(5Arabis pauciflora [Grimm] Garcke) (Jena, Germany) . . . . .
Lepidium campestre L.
(Lengerich, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Matthiola incana (L.) R. Br.
(Bot. Garden Jena, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rorippa amphibia (L.) Bess.
(near Cologne, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sinapis alba L.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a
Apetala3
Chs
% GC CONTENT (length in bp)a
Apetala3
Chs
Not sequenced
AF249000
33.6 (401)
Not sequenced
AL132971
U30729
AF248989
Not sequenced
Not sequenced
Not sequenced
AF248988
AF248971
Not sequenced
33.0 (409)
AF248972
AF248995
33.7 (412)
AF248984
Not sequenced
34.0 (535)
AF248981
Not sequenced
37.6 (506)
AF248973
AF248974
AF248975
Not sequenced
33.8 (486)
33.8 (485)
33.0 (421)
Not sequenced
AF248994
AF248982
AF248996
36.7 (474)
37.8 (471)
AF248978
AF043610
AF249991
Not sequenced
35.0 (528)
34.8 (1,036)
37.5 (407)
AF248980
Not sequenced
34.4 (599)
Not sequenced
AF248993
AF248983
AF248987
35.0 (548)
35.5 (529)
AF248985
AF248986
34.0 (551)
36.4 (499)
Not sequenced
AF248999
AF248970
Not sequenced
32.2 (430)
AF248979
AF248998
36.4 (513)
39.4 (412)
AF248976
AF248990
34.4 (508)
34.5 (437)
AF248977
AF248997
36.9 (407)
37.8 (409)
AF248968
AF248969
Not sequenced
AF248992
Not sequenced
X16437
34.8 (520)
35.1 (447)
38.4 (396)
28.5 (1,111)
34.6 (529)
34.6 (529)
34.1 (508)
33.1 (556)
34.9 (540)
36.7 (401)
30.8 (392)
29.7 (664)
The average GC contents of the Apetala3 and Chs promoter regions are 34.7% (sn21 5 1.3) and 34.9% (sn21 5 3.1), respectively.
alignment which we judged to be highly reliable were
used for phylogenetic analysis. These parts showed pairwise similarities .50% and were not disrupted by extensive microsatellite sequences. We excluded DNA regions which were used as primer-binding sites for amplification. Phylogenetic distances were computed using
Kimura’s (1980) two-parameter model, and the resulting
distance matrices were subjected to the neighbor-joining
algorithm as implemented in TREECON (version 1b;
Van de Peer and De Wachter 1997), which was also used
to perform bootstrap analysis (Felsenstein 1985) with
1,000 replicates.
Results
DNA Sequence Variation in the Chs Promoter Regions
The Chs promoter fragments we obtained varied in
length from 392 bp in Cochlearia pyrenaica to 1,111
bp in Arabidopsis griffithiana (table 1). No allelic variation was detected within species. The mean G/C con-
Promoter Analysis in Cruciferous Plants
Table 2
Overview of the Amplification Results of the Apetala3
Promoter Region with Different Primer Combinations
prom1.for/ prom2.for/ prom3.for/
genapet.rev genapet.rev genapet.rev
Arabis alpina AFRICA . . . . .
A. alpina EUROPE . . . . . . . .
Arabis drummondii. . . . . . . . .
Arabis glabra . . . . . . . . . . . . .
Arabis hirsuta . . . . . . . . . . . . .
Arabis turrita . . . . . . . . . . . . .
Arabidopsis thaliana COL. . .
A. thaliana LER . . . . . . . . . . .
Barbarea vulgaris. . . . . . . . . .
Capsella rubella . . . . . . . . . . .
Cardaminopsis petraea . . . . .
Cardaminopsis halleri . . . . . .
Cochleria pyrenaica . . . . . . . .
Fourraea alpina . . . . . . . . . . .
Lepidium campestre . . . . . . . .
Matthiola incana . . . . . . . . . .
Rorippa amphibia. . . . . . . . . .
2
2
1
2
1
2
1
1
1
1
1
1
2
1
1
2
1
2
2
1
2
1
1
1
1
1
2
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
NOTE.—Primer positions are indicated in figure 2 (for primer sequences,
refer to Material and Methods. 1 5 positive PCR reaction; 2 5 no PCR product.
tent was 34.9% (SD 5 3.1%), with a minimum of 28.5%
in the long A. griffithiana promoter fragment. The highest G/C content was found in the short promoter fragment of Fourraea alpina, with 39.4% (table 1). The
alignment of the entire promoter region is shown in figure 1. At alignment position 121, we removed from consideration DNA sequences which were not alignable.
These fragments were mostly shorter than 75 bp, but in
the case of A. griffithiana (657 bp) and S. alba (298 bp),
they were much longer.
In our earlier investigation of Chs coding sequences, we used a forward primer located in the ACE-MRE
region (CHS-FOR1 in Koch, Haubold, and Mitchell-
1885
Olds 2000) at alignment position 496 (fig. 1, supplementary material). DNA sequence comparisons with
these data demonstrated that our promoter fragments
corresponded to the previously isolated Chs genes,
which have been shown to be orthologous (Koch, Haubold, and Mitchell-Olds 2000). Therefore, our corresponding promoter fragments represented exclusively
orthologous DNA regions.
DNA Sequence Variation of Apetala3 Promoter
Regions
The sequencing of different subfragments of the
Apetala3 promoter region (table 2) mostly resulted in
overlapping fragments of identical sequences. In the
case of Rorippa amphibia with the primer combination
APET3.for/APET.rev, one additional sequence was
found. In Arabis hirsuta, we detected three different
genes corresponding to the three primer combinations.
However, A. hirsuta is an autotetraploid plant, so we
probably isolated alleles from the two duplicated loci.
The alignment of the entire promoter region is
shown in figure 2 (supplementary material). The length
of the fragment (APET1.for/APET.rev) varied from 486
bp in R. amphibia to 1,036 bp in B. oleracea. The mean
G/C content was 34.7% (SD 5 1.3%), with a minimum
of 32.2% in the C. pyrenaica promoter fragment. The
highest G/C content was found in the promoter fragment
of A. glabra, with 37.6% (table 1). At alignment position 62, we found a 459-bp indel in the B. oleracea
promoter.
Conserved Elements in the Chs Promoter Region
Several conserved elements and regions could be
detected among the different Chs promoters (fig. 1, supplemetary material). A G-box motif described previous-
FIG. 1.—Expression levels of Chs promoter fragments in an Arabidopsis thaliana protoplast transient expression system. Black bars show
the dark control.
1886
Koch et al.
learia excelsa. A CAAT promoter consensus sequence
has been found at positions 375 and 407. The sequence
is highly conserved and might be responsible for tissuespecific promoter activity (Shirsat et al. 1989).
A consensus GT-1-binding site (GAAAAA) was
found at position 452. This motif is present in many
light-regulated genes from numerous species (Villain,
Mache, and Zhou 1996; Zhou 1999). A related
GGTTAAA/TA/T motif has been described by Lawton et
al. (1991) in a silencer region of Chs from the bean.
Several other highly conserved regions (.50% invariable nucleotide positions in a fragment a minimum of
15 bp in length) have no known functional role and were
labeled as ‘‘region 1’’ and ‘‘region 2’’ (fig. 1, supplementary material; positions 439–555 and 471–488, covering the GT-1-binding site). These conserved sequence
motifs may represent novel regulatory elements.
Conserved Elements in the Apetala3 Promoter Region
FIG. 2.—Schematic phylogenetic relationships of taxa under study
derived from coding matK, Chs, and Adh sequence data (Koch, Haubold, and Mitchell-Olds 2000, 2001). Statistically not–highly-supported nodes of Matthiola and Lepidium are highlighted by dashed lines.
ly by Hartmann et al. (1998) was located at alignment
position 13. Because this motif was part of the amplification primer APET3.for, we were not able to detect
mutations at these sites. Amplification worked in all
taxa, and hence this element is presumably ubiquitous.
An additional G-box was located at position 270. This
G-box was only present in the promoters of Arabis jaquinii and Arabis alpina, which form a monophyletic
group (Koch, Bishop, and Mitchell-Olds 1999; Koch,
Haubold, and Mitchell-Olds 2000, 2001). The third Gbox was located at position 507, in close proximity to
the TATA box (position 604). It has previously been
described as a highly conserved promoter region in crucifers, Phaseolus vulgaris (Faktor et al. 1997b), Petrosilinum crispum (Schulze-Lefert al. 1989a, 1989b;
Block et al. 1990), Gentiana triflora (Kobayashi et al.
1998), and Petunia hybrida (Van der Meer et al. 1990).
This G-box motif is also referred to as the ACGT-containing element (ACE; Hartmann et al. 1998). In close
39 proximity at position 539 there was an H-box motif
(Loake et al. 1992; Faktor et al. 1997b), which has also
been denoted the Myb recognition element (MRE) motif
(Hartmann et al. 1998). Between the MRE and the ACE
elements there was an A-box motif, which was first observed in phenylalanine ammonia-lyase (PAL) in P.
crispum (Logemann, Parniske, and Hahlbrock 1995).
Another H-box-like (or MRE) motif was located at
position 397. Database comparisons with PLACE, a collection of promoter elements (http://www.dna.affrc.go.
jp/htdocs/PLACE/signalscan.html; Higo et al. 1999) revealed several other conserved cis-acting regulatory elements with unknown functions in the Chs promoter
region. There was an A-box motif (TACGTA) at position 220, related to the G-box or MRE with a core
ACGT sequence. However, this ACGT sequence only
appeared in the distantly related R. amphibia and Coch-
Several MADS-domain-containing proteins bind
DNA at a CC(A/T)6GG core consensus binding site
which has been referred to as the CArG box (SchwarzSommer et al. 1992; Wynne and Treisman 1992; Huang,
Mizukami, and Ma 1993; Shiraishi, Okada, and Shimura
1993; Hill et al. 1998). The Apetala3 promoter of A.
thaliana contains three CArG box motifs (Hill et al.
1998), which may serve as binding sites for one or more
MADS-domain-containing proteins (Irish and Yamamoto 1995). These boxes are highly conserved among
the cruciferous plants analyzed in this study and are
identified in figure 2 (supplementary material) at positions 428, 476, and 500.
Several other highly conserved regions were located exclusively upstream of the CArG1 motif, and none
of them have been characterized experimentally. (As for
the analyzed Chs-promoter region, we defined regions
as ‘‘highly conserved’’ if more than 50% of a DNA
sequence with a minimum length of 15 bp was invariable.) However, strong evidence for functional relevance
of promoter regions upstream of the CArG1 motif is
provided by experimental analysis of the Apetala3 promoter in A. thaliana (Hill et al. 1998).
The Apetala3 alignment (fig. 2, supplementary material) shows four conserved regions, which we designated region 1 (alignment positions 73–133), region 2
(alignment positions 216–230), region 4 (alignment positions 365–381), and duplicated region 3a (alignment
positions 328–347) and region 3b (alignment positions
383–402). In each of regions 1, 2, and 4, there was one
MYB-binding site (with a consensus motif CNGTTR;
Urao et al. 1993). Only in the highly conserved regions
3a and 3b (90% invariable sites) were we unable to identify known regulatory motifs.
Evidence for additional motifs is ambiguous. At
position 78 there was a second putative MYB–homologbinding site, previously recognized in maize (consensus
motif CCWACC; Grotewold et al. 1994). Overlapping
with this myb element was a sequence similar to a region
necessary for circadian expression of the tomato Lhc
(light harvesting complex) gene (CAANNNNATC; Pie-
Promoter Analysis in Cruciferous Plants
FIG. 3.—Neighbor-joining distance tree based on Chs promoter
sequences. Bootstrap support is given along the branches.
chulla, Merforth, and Rudolph 1998), and overlapping
with the 39 end of this putative myb element was a Gbox-like CACCTG motif. At positions 73 and 243 there
were also core sites (AAAG) required for binding of Dof
proteins (DNA-binding proteins) in maize, with presumably only one zinc finger (Yanagisawa and Schmidt
1999). Interestingly, a few base pairs upstream of this
AAAG recognition sequence, there was an ACTTTA
motif (alignment position 53), which has been shown to
act as a Dof–protein-binding site (Baumann et al. 1999).
We also found the same motif at alignment position 421
close to the CArG1 box.
Phylogenetic Analysis
With the exception of Lepidium campestre and M.
incana, the evolutionary relationships of these species
have been considered in previous studies (Koch, Haubold, and Mitchell-Olds 2000, 2001). A schematic phylogenetic network based on matK and Chs coding regions considering only taxa analyzed herein is redrawn
from Koch, Haubold, and Mitchell-Olds (2001) in figure
2 for further comparisons. The phylogenetic tree estimated from Chs promoter sequence divergence values
(fig. 3) differs from the combined matK/Chs tree (fig. 2)
only in the relative positions of L. campestre and M.
incana. However, as outlined above, there is little statistical support (bootstrap values and decay indices in
Koch, Haubold, and Mitchell-Olds 2001) for the relative
positions of Lepidium and Matthiola, and therefore the
conflict among these differing topologies is statistically
insignificant. This means that Chs promoter sequence
evolution corresponds closely with Chs gene coding sequences as well as several other genes and DNA regions. This holds at least for the selected portions of the
sequences for which a reliable alignment was found.
However, distance analysis of the total alignment resulted in the same tree topology with some extremely
long branches. Because it is not possible to calculate
1887
FIG. 4.—Neighbor-joining distance tree based on Apetala3 promoter sequences. Bootstrap support is given along the branches.
simple synonymous or nonsynonymous mutation rates
for pairwise comparisons, we used the Kimura (1980)
two-parameter distances to compare Chs promoter regions (alignable regions as indicated in fig. 1, supplementary material) with the corresponding Chs coding
regions (Koch, Haubold, and Mitchell-Olds 2000).
These data showed that within the selected promoter regions the mean substitution rate in the Chs promoter
region is 0.58 times as low (SD 5 0.18) as the mean
synonymous substitution rate in the corresponding coding Chs region.
The phylogenetic topology from the Apetala3 promoter sequences is nearly identical to that obtained from
the Chs promoter region (fig. 4). We take this as evidence that we analyzed orthologous promoter fragments
of the Apetala3 MADS-box regulatory gene. It has recently been shown that there is a large monophyletic
assemblage that includes the major floral homeotic gene
groups AGAMOUS, Apetala3, PISTILLATA, and APETALA1/AGL9, and these four MADS-box genes were
clearly separated from each other in previous phylogenetic analyses (Purugganan et al. 1995; Purugganan
1997). Therefore, it is unlikely that we analyzed promoter fragments not related to the Apetala3 gene. Three
Apetala3 coding regions have been reported (Brassica
napus AF124814, A. thaliana D21125, and Cardaminopsis petraea AF143380). For these three taxa, the
mean substitution rate in the apetala3 promoter regions
selected for phylogenetic analysis is 0.59 (SD 5 0.17)
times as low as the synonymous substitution rate in the
corresponding coding regions.
Chs Promoter Expression Analysis
Functional analysis of the Chs promoter fragments
from R. amphibia, F. alpina, A. griffithiana, C. petraea,
A. alpina, and Aethionema grandiflora revealed no major differences in UV light response. Absolute expression levels varied at most by a factor of two among taxa
1888
Koch et al.
(fig. 1), and Chs induction varied from 75- to 204-fold.
Error bars represent standard deviation from eight transfection experiments. These data show that the conserved
sequences detected in the various promoters are functional and that variable sites do not interfere with functionality. Our results indicate that conserved promoter
elements do indeed have a regulatory function and that
sequence comparisons can be used to infer the type and
location of important cis-acting elements in promoter
sequences. Without additional information, it is still difficult to relate these elements with the response to potential external or internal stimuli. However, the increasing information from parallel expression analysis (Zhu
and Wang 2000) will soon provide the data required for
inferring such functional relationships.
H-box-like sequence at alignment position 396 (fig. 1,
supplementary material).
Recently, Seki et al. (1996) presented an overview
of these elicitor-responsive cis elements and clearly
showed similarities among Chs and PAL gene promoters
from different species. Interestingly, only a few additional regions have been subjected to functional dissection analysis. Nuclear factors (SBF-1) have been identified in P. vulgaris that bind to SBF-1 boxes in the 59
region of the bean Chs15 gene promoter regulating tissue-specific expression (Hotter et al. 1995). These SBFboxes contain a consensus motif identical to the GT-1
recognition motif. We found a highly conserved GT1like motif (fig. 1, supplementary material) at alignment
position 452 which might have a similar function.
Discussion
Functional Dissection of the Apetala3 and Chs
Promoters
Perspective
Hill et al. (1998) reported functional dissection of
the Apetala3 promoter region in A. thaliana, and identified regions of promoter sequence conservation between A. thaliana and B. oleracea. However, the results
of their Arabidopsis-Brassica sequence comparison differed substantially from our results, which are based on
16 sequences from cruciferous plants. Only a few elements, such as the CArG motif and region 4, were apparent in the comparison of Hill et al. (1998). The remaining highly conserved motifs (e.g., regions 1, 2, and
3) were not apparent in that study of only two species.
Nevertheless, Hill et al.’s (1998) experiments clearly
demonstrated that a promoter region covering our region
1 (fig. 2, supplementary material) is required for antherspecific expression. This work indicates that our defined
promoter regions 3a, 4, and 3b are required for early
(stages 3–5) and for petal-specific expression during floral development, while region 2 is required for expression in the filaments. Hill et al. (1998) concluded that
specific temporal and spatial cis-acting elements exist
within the Apetala3 promoter. These predicted elements
correspond to the conserved regions we identified in this
study.
Hartmann et al. (1998) showed that a light regulation unit (LRU) containing the ACE and the MRE was
sufficient for UV/blue light-regulated expression of Chs.
The corresponding LRU from bean Chs15 has been intensively studied (Arias, Dixon, and Lamb 1993), and it
has been shown that the G-box (ACE) and the H-box
(MRE) make major contributions to the transcription of
the Chs15 promoter in vivo. Combinatorial specificity
of these elements has also been found in promoters of
genes encoding phenylpropanoid biosynthetic enzymes
(Lois et al. 1989; Leyva et al. 1992). The corresponding
LRU regions in Chs promoters of the French bean (Faktor et al. 1996) and the petunia (van der Meer et al.
1990) are also responsible for tissue-specific activity. Finally, additional H-boxes binding MYB classes of transcription factors are present upstream of the LRU unit
of bean Chs15 (Arias, Dixon, and Lamb 1993). This
corresponds to our observation of a highly conserved
We analyzed interspecific patterns of sequence conservation in promoter regions of two genes, Chs and
Apetala3. Our results show that known functionally important regulatory elements are conserved among crucifer relatives of Arabidopsis (figs. 1 and 2, supplementary material). Other, previously unknown, elements
were identified which are even more highly conserved
than the known motifs which have been identified by
functional studies. In addition, we showed that Chs promoter sequences from a broad sample of related species
are sufficient for light-regulated gene expression in transient expression assays in Arabidopsis.
Comparative analyses of putative regulatory regions are increasingly important in genomics. Sequences
of CFTR, the gene responsible for cystic fibrosis in humans, have been compared in humans, mice, and Fugu
(Davidson et al. 2000; Ellsworth et al. 2000). Humanmouse comparisons found many intergenic and intron
segments with high levels of conservation, providing little ability to identify which regions were functionally
important in gene regulation. With greater evolutionary
divergence, genomic sequence 59 of CFTR is highly divergent between pufferfish and mammals, except for
conserved putative CRE and CAAT box elements. Likewise, human-mouse comparison of the SNCA gene,
which may influence development of Alzheimer’s and
Parkinson’s diseases, identified a novel 59 element that
regulates normal expression in transient assays (Touchman et al. 2001). Wasserman et al. (2000) studied conserved promoter regions from 28 human-mouse pairs of
skeletally expressed genes and identified a number of
known and inferred enhancer elements.
Comparative analysis of promoter sequences from
related species is a powerful tool to identify conserved
and putatively functional elements. Rapidly accumulating knowledge about the function and regulation of
genes coming from the model plant A. thaliana can be
easily transferred to its closest relatives. Wild relatives
provide a broad spectrum of naturally occurring genotypes and phenotypes, which are presumably adapted to
a range of environments and natural histories. This will
permit hypothesis testing about the evolutionary significance of character change and suites of traits. Well-
Promoter Analysis in Cruciferous Plants
supported phylogenies of numerous Brassicaceae and
estimates of divergence times provide an important resource for these studies (Koch, Haubold, and MitchellOlds 2000, 2001). These can be used to compare rates
of evolution. In our analysis, the greatest evolutionary
divergence was about 45 Myr (for the Chs promoter of
A. thaliana compared with A. grandiflora).
Bioinformatic analyses have used three approaches
to identify putative regulatory regions within promoters:
pattern recognition, phylogenetic footprinting among
several species, and within-species comparison of promoters from coregulated genes (Fickett and Wasserman
2000; Wasserman et al. 2000). Phylogenetic footprinting
is particularly important, because it reduces the search
space for subsequent computational analysis. In addition, comparison of promoter sequences from several
species at different levels of evolutionary divergence
may provide improved identification of putative functionally important sites (e.g., Davidson et al. 2000).
Likewise, expression profiling can identify groups of
coregulated genes which may display above-average frequencies of particular regulatory motifs (Maleck et al.
2000). All three approaches will be useful for understanding gene regulation in A. thaliana.
Despite successful comparisons in our experiments
and in other studies, regulatory function may be maintained even in the absence of sequence conservation. For
example, the Drosophila melanogaster and Drosophila
pseudoobscura promoters of even-skipped mediate conserved patterns of stripe 2 expression during embryo
development. In contrast, the functionally important
stripe 2 enhancer shows considerable divergence between these two species. Chimeric promoters (containing both combinations of the 59 and 39 halves of the D.
melanogaster and D. pseudoobscura upstream regions)
are no longer expressed in the wild-type pattern (Ludwig
et al. 2000). This decoupling of conservation and function will complicate attempts to identify functionally important regulatory motifs by comparative genomics.
Supplementary Material
Sequence alignments are provided on the Molecular Biology and Evolution website.
Acknowledgments
We thank Domenica Schnabelrauch and Antje Figuth for help with the sequence analysis. This work was
supported by the Max-Planck-Gesellschaft, and by
grants to T.M.-O. from the U.S. National Science Foundation (DEB-9527725) and the European Union.
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JULIAN ADAMS, reviewing editor
Accepted June 12, 2001